T-786C polymorphism of the NOS-3 gene and the endothelial cell response to fluid shear stress-a proteome analysis.

Endothelial dysfunction is a common denominator of cardiovascular disease. Central to endothelial dysfunction is a decrease in the bioavailability of nitric oxide (NO) synthesized by endothelial NO synthase (NOS-3). In vivo, the level of fluid shear stress (FSS) exerted by the flowing blood determines NOS-3 expression. However, in contrast to the -786T variant of the nos-3 gene, the -786C variant is not sensitive to shear stress. Consequently, cells homozygous for this variant have an inadequate capacity to synthesize NO. Therefore, we have compared shear stress-induced protein expression in human primary cultured endothelial cells with TT or CC genotype. Cells with the CC genotype exhibited a greatly reduced FSS-induced NOS-3 expression as well as a diminished NO synthesis capacity when compared to TT genotype cells. Proteome changes in response to FSS (30 dyn/cm(2) for 24 h) were monitored by 2D-gel electrophoresis/densitometry/mass spectrometry. Of a total of 14 FSS-sensitive proteins, 8 were identically expressed in all cells. Four proteins, all of them part of the NO-dependent endoplasmic reticulum-stress response, were up-regulated by FSS only in cells with TT genotype. In contrast, CC genotype cells responded to FSS with a unique increase in manganese-containing superoxide dismutase expression. These differences in protein expression may (i) reflect the low bioavailability of NO in cells homozygous for the -786C variant of the nos-3 gene and (ii) point to a mechanism by which this deficit is counterbalanced by protecting the less abundant NO from rapid degradation.

Differential proteomic analysis of lymphocytes treated with mycophenolic acid reveals caspase 3-induced cleavage of rho GDP dissociation inhibitor 2.

The antiproliferative immunosuppressive drug mycophenolic acid (MPA) is an uncompetitive inhibitor of inosine monophosphate dehydrogenase, a key enzyme in de novo synthesis of purine nucleotides. The latter are not only required for synthesis of DNA and RNA but also are essential for the regulation of numerous cellular signaling pathways modulated by guanine nucleotide binding proteins (G proteins). We undertook an analysis of the influence of MPA on protein expression in a T-lymphoblast cell line (CCRF-CEM), which displays concentration-dependent inhibition of proliferation by MPA to obtain insight into the influence of MPA on the cellular proteome. Cells were stimulated with phorbol myristate acetate/ionomycin and incubated in the presence or absence of MPA. Two-dimensional electrophoresis and densitometric imaging revealed 11 differentially expressed protein spots (P < 0.05) on MPA treatment, 6 with increased and 5 with decreased abundance. After in-gel tryptic digestion, proteins were identified by quadrupole time-of-flight mass spectrometry. Proteins displaying increased abundance after MPA treatment included splicing factor arginine/serine-rich 2, prostaglandin E synthase 3, peptidyl-prolyl cis-trans isomerase A, and deoxyuridine 5'-triphosphate nucleotidohydrolase. Endoplasmin, proliferating cell nuclear antigen, acidic leucine-rich nuclear phosphoprotein 32 family member A, and cofilin 1 showed decreased abundance after MPA treatment. Three separate spots (1 decreased and 2 increased abundance) were identified as Rho guanosine diphosphate dissociation inhibitor 2 (Rho GDI 2) proteins. Western blotting with a monoclonal antibody directed against the Rho GDI 2 site cleaved by caspase 3 demonstrated 1 spot with increased abundance to be the caspase 3-cleaved product of Rho GDI 2 lacking the first 19 amino acids. Rho GDI 2 plays a central regulatory role in the activation of Rho guanosine triphosphatases that function as molecular switches in cell signaling pathways affecting cell cytoskeletal dynamics and motility. Our data suggest that MPA can modulate Rho GDI 2 levels in T lymphocytes, thereby potentially disrupting cell signaling pathways important for T-cell function.

Everolimus exposure in cardiac transplant recipients is influenced by concomitant calcineurin inhibitor.

In two separate pharmacokinetic studies, the drug interaction between immunosuppressive agents was examined in a total of 12 cardiac transplant recipients by conversion of the concomitant immunosuppressant. In six patients under continuous tacrolimus therapy, the concomitant drug azathioprine was converted to everolimus (PK-TAC study). No significant effect on tacrolimus pharmacokinetic parameters was observed. In the second study in which the patients were converted from cyclosporine to tacrolimus under continuous everolimus therapy (PK-EVL study), a significant decrease in everolimus predose concentration (from 4.2 to 2.3 microg/L), maximum concentration (from 9.1 to 5.9 microg/L), and area under the concentration time curve (mean values decreased from 64.2 to 33.7 microg*h/L) was found, indicating a lower everolimus exposure. A pharmacokinetic interaction between cyclosporine and everolimus has been described previously for healthy volunteers after single-dose application and presumably originates from a comparatively greater inhibition of hepatic CYP3A4 or P-glycoprotein efflux transporter with a low-dose cyclosporine regimen. Our results confirm this interaction under clinical conditions and suggest close drug monitoring when converting the calcineurin inhibitor under concomitant mammalian target of rapamycin-inhibitor therapy.

Real-time assessment of hepatic function is related to clinical outcome in critically ill patients after polytrauma.

OBJECTIVES: The aim was to investigate the outcome MODS/MOF in critically ill patients with regard to early hepatic dysfunction. METHODS: Thirty adult polytrauma patients admitted to the ICU, with ISS >or=16 were prospectively investigated. Real-time liver function was assessed using the MEGX test and arterial ketone body ratio (AKBR) 12-24 h after admittance to ICU, and on days 3, 5, 8, 12. RESULTS: Six patients (19%) died between days 4 and 29. Non-survivors were older (64.2 vs. 31.5 years), had a significantly higher ISS (40.5 vs. 30; p=0.002) and MODS score (9.5 vs. 5; p=0.001) on admittance to the ICU than survivors. On day 3 MEGX values (31 vs. 71.3 microg/L; p=0.001) and the AKBRs (0.6 vs. 1.3; p=0.001) were significantly lower in non-survivors than in survivors whereas IL-6 levels were significantly higher in the former group (519 vs. 61 microg/L; p=0.05). CONCLUSIONS: The MEGX test and AKBR are sensitive early indicators of hepatic dysfunction in severely injured polytrauma patients at risk for developing MODS/MOF.

Pharmacokinetics of mycophenolate mofetil in hematopoietic stem cell transplant recipients.

Mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), is increasingly used in the prophylaxis of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HCT). Few pharmacokinetic data are available about the use of MMF for this indication. This case series aimed at analyzing the pharmacokinetics of MMF in a population of HCT recipients representative for everyday practice. From 15 HCT recipients, serial plasma samples were taken after twice-daily oral intake of MMF. Plasma concentrations of total MPA and its glucuronide metabolites, as well as free MPA, were quantified. Median apparent oral MPA clearance (CL/F), apparent half-life, and total MPA area under the curve for hours 0 to 12 (AUC0-12, normalized to 1000 mg MMF) were, respectively, 56 L/h (range: 29-98 L/h), 2.3 hours (range: 0.8-5.7 hours), and 18.0 mg*h/L (range: 10-35 mg*h/L). Total MPA concentrations were below 2 mg/L 8 hours after MMF administration, indicating reduced enterohepatic recirculation. Median free MPA AUC0-12 (normalized to 1000 mg MMF) was 224 microg*h/L (range: 56-411 microg*h/L). Because of high CL/F, total MPA exposure in HCT recipients is low and apparent half-life is short in comparison with reference values from renal transplantation. Exposure may be improved in HCT recipients by higher or more frequent MMF dosing.

Quantification by liquid chromatography tandem mass spectrometry of mycophenolic acid and its phenol and acyl glucuronide metabolites.

BACKGROUND: We developed and validated a rapid and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) procedure for the quantification of mycophenolic acid (MPA) and its phenol glucuronide (MPAG) and acyl glucuronide (AcMPAG) metabolites. METHODS: We performed protein precipitation on all samples (calibrators, quality controls, and patient samples) and then subjected them to online solid-phase extraction followed by reversed-phase liquid chromatography for 4.0 min. The carboxybutoxy ether of MPA (MPAC) was used as the internal calibrator. The separated compounds (MPA, MPAG, AcMPAG, and MPAC) were detected by electrospray ionization-coupled MS/MS. We compared LC-MS/MS results with results for the same samples obtained with a validated HPLC procedure with an ultraviolet detector. RESULTS: Comparison with the validated HPLC-ultraviolet procedure demonstrated good agreement. The Passing-Bablok regression was y = 0.968x - 0.058 for MPA, y = 1.08x - 1.697 for MPAG, and y = 0.952x + 0.076 for AcMPAG. Assay imprecision showed a CV <10% at 3 concentrations for each compound. The lower limit of quantification was 0.1 mg/L for MPA, 1.0 mg/L for MPAG, and 0.05 mg/L for AcMPAG. The mean analytical recovery was 90%-110%. The assay was linear from 0.1 to 50 mg/L for MPA (r = 0.9987), from 1 to 500 mg/L for MPAG (r = 0.9999), and from 0.05 to 10 mg/L for AcMPAG (r = 0.9988). Quantification of the compounds was not affected by in-source fragmentation or ion suppression. CONCLUSION: The LC-MS/MS assay described here is valid and reliable for the quantification of total MPA, MPAG, and AcMPAG in serum.

Differential expression of major histocompatibility complex class I molecules in the brain of a New World monkey, the common marmoset (Callithrix jacchus).

It has been supposed that central nervous neurons do not express MHC class I molecules. However, recent studies clearly demonstrated functional MHC class I expression in the rodent brain. In the present study, we have extended these studies and investigated the presence of MHC class I transcripts and proteins in the brain of a non-human primate species, the common marmoset monkey (Callithrix jacchus). Using in-situ hybridization, we found strong expression of MHC class I transcripts in neocortex, hippocampal formation, substantia nigra and nucleus ruber. In-situ hybridization with emulsion autoradiography demonstrated MHC class I mRNA in distinct pyramidal neurons of cortex and hippocampus, in granule neurons of the dentate gyrus, in dopaminergic neurons of substantia nigra and in motor neurons of nucleus ruber. Immunocytochemistry confirmed MHC class I protein expression in these neurons. Two monoclonal antibodies, MRC-Ox18 and HB115, reacted differentially with MHC class I proteins on neuronal and non-neuronal cells, respectively. Interestingly, in marmoset monkeys that were immunosuppressed with FK506 (tacrolimus), expression of neuronal MHC class I proteins, which could be detected with MRC-Ox18, was either very low (neocortex, nucleus ruber, substantia nigra) or absent (hippocampus). In contrast, class I expression in endothelial cells, which was detected by HB115, was not affected by immunosuppression. Our data show that selected neurons in the brain of a non-human primate express MHC class I molecules and that this expression can be modulated by immunosuppression.

Pharmacokinetics and bioavailability of mycophenolic acid after intravenous administration and oral administration of mycophenolate mofetil to heart transplant recipients.

The aim of this prospective study was to characterize the multiple-dose pharmacokinetics of mycophenolic acid (MPA) after administration of a 3-hour intravenous (IV) infusion of mycophenolate mofetil (MMF, CellCept) at a dose level of 1.5 g every 12 hours for 5 full days to cardiac allograft recipients and to compare the bioavailability of MPA after a switch from the IV infusion to an oral dose of 1.5 g every 12 hours from day 6. In addition to MMF, patients received cyclosporine and prednisolone. Blood (EDTA) samples for full pharmacokinetic profiles were obtained for 9 patients on days 3 and 5 (IV MMF) and on days 6 and 10 (oral MMF). They were centrifuged within 45 minutes of collection, and plasma was stabilized by addition of ortho-phosphoric acid to prevent in vitro conversion of MMF to MPA. Plasma concentrations of MPA were determined using a validated HPLC procedure. The median MPA AUC on day 6 (29.7 mg.h/L) after the first oral dose was slightly lower than the AUCs on the other study days (34.2, 33.8, and 33.8 mg.h/L on days 3, 5, and 10, respectively). Pairwise comparison of the individual days revealed statistically significant (P<0.05) differences between day 6 and day 3 and between day 5 and day 3. The Cmax on day 6 was significantly lower than that on study days 3 and 5. The bioavailability of MPA from the oral MMF formulation was estimated as the ratio of the AUC on day 6 or 10 to the AUC on day 5 when steady state was presumed to have been reached with the IV formulation. The mean ratios (expressed as percentage) for the log-transformed AUCs were 91.6% and 107.8% on days 6 and 10, respectively, relative to day 5. The 90% confidence interval (CI) on day 6 (79.3% to 105.8%) was marginally below the range (80%-125%) required to conclude that the formulations are bioequivalent, whereas on day 10 the 90% CI (93.3% to 124.7%) was within this range. In the case of the Cmax values, however, the 90% confidence intervals fell outside of this range (day 6, 57.2% to 92.8%; day 10, 70.6% to 114.9%). The results of this study show that heart transplant recipients receiving the IV formulation of MMF (1.5 g BID) are not subject to a greater drug exposure than that seen with the oral formulation (1.5 g BID) and that the oral MMF formulation shows excellent, high, and consistent bioavailability (mean 95%) based on comparison with the IV formulation.

Mycophenolate mofetil in organ transplantation: focus on metabolism, safety and tolerability.

Mycophenolate mofetil (MMF) received its first approval for the prevention of renal allograft rejection in 1995 and has now become the most frequently used antiproliferative agent in maintenance immunosuppressive therapy for kidney, pancreas, liver and heart transplantation. In addition, its use for the treatment of autoimmune diseases steadily increases. This review focuses on the miscellaneous pharmacodynamic properties of the drug, its pharmacokinetics in healthy subjects, recipients of different organ transplants and combination therapy with other pharmaceuticals, as well as its safety profile. The immunosuppressive activity of MMF is thought to derive mainly from the potent and selective inhibition of purine synthesis in both T and B lymphocytes. In contrast to other immunosuppressants on the market, it is metabolised primarily by glucuronidation and lacks nephrotoxicity, cardiovascular toxicity or diabetogenic potential, thus making it a suitable candidate for combination regimens. The most important side effects under MMF include gastrointestinal disorders, of which the underlying mechanisms are not yet fully understood, but seem to be complex and related to both effects of mycophenolic acid and its acyl glucuronide, as well as to decreased -immunity due to general immunosuppression after transplantation.

Validation of a rapid and sensitive liquid chromatography-tandem mass spectrometry method for free and total mycophenolic acid.

BACKGROUND: Because mycophenolic acid (MPA) is highly protein bound and because the free fraction is the pharmacologically active portion, a rapid, reliable, and sensitive procedure is required to study the relationship between free MPA and treatment efficacy/toxicity. Liquid chromatography-tandem mass spectrometry is ideally suited for such a method. METHODS: Free MPA was isolated from plasma by ultrafiltration. An online extraction cartridge with a column-switching technique, analytical liquid chromatography over an Aqua Perfect C(18) column, and electrospray tandem mass spectrometry was used to quantify free and total MPA. To investigate ion suppression, a continuous infusion of MPA was introduced into the effluent from the HPLC column, and different ultrafiltrates and extracted plasma samples were injected on the column. RESULTS: A chromatographic run time of 4 min separated MPA from metabolites and internal standard, thereby avoiding interference from in-source fragmentation. Ion suppression occurred well before elution of MPA and internal standard. The lower limit of quantification for free MPA was 0.5 microg/L, and the method was linear to 1000 microg/L. Interassay imprecision (CV) was <10% for free MPA (0.5-333 microg/L). Agreement was good for free MPA (n = 52) and total MPA (n = 106) between the proposed method and a validated HPLC method with ultraviolet detection. The Passing-Bablok regression line was: y = 0.95x + 0.27 microg/L for free MPA and y = 0.98x + 0.03 mg/L for total MPA. CONCLUSIONS: The presented method allows the accurate, precise, and rapid determination of free and total MPA in plasma over a wide analytical range covering the concentrations relevant to pharmacokinetic studies and routine monitoring of this drug.

Differences in nucleotide hydrolysis contribute to the differences between erythrocyte 6-thioguanine nucleotide concentrations determined by two widely used methods.

BACKGROUND: Measurement of 6-thioguanine nucleotide (6-TGN) concentrations in erythrocytes is widely accepted for use in optimization of thiopurine therapy. Various chromatographic methods have been developed for this purpose. In preliminary experiments we observed a considerable difference between 6-TGN concentrations determined with two widely used methods published by Lennard (Lennard L. J Chromatogr 1987;423:169-78) and by Dervieux and Boulieu (Dervieux T, Boulieu R. Clin Chem 1998;44:551-5). We therefore investigated methodologic differences between the two procedures with respect to hydrolysis of 6-TGNs to 6-thioguanine (6-TG) in more detail. METHODS: We analyzed 6-TGNs in erythrocyte preparations (n = 50) from patients on azathioprine therapy by both methods, using the original protocols. In one set of experiments, we replaced the 0.5 mol/L sulfuric acid in the Lennard method with the 1 mol/L perchloric acid used by Dervieux and Boulieu. In a second set of experiments, we investigated the effect of various dithiothreitol (DTT) concentrations on 6-TG recovery with both methods. In a third set of experiments, we determined the effect of hydrolysis time on both protocols. RESULTS: Direct comparison of both methods showed that 6-TGN concentrations were, on average, 2.6-fold higher in the Dervieux-Boulieu method over the concentration range tested, although the correlation (r = 0.99; P <0.001) was good. Replacement of sulfuric acid by perchloric acid reduced this difference to approximately 1.4-fold (r = 0.99; P <0.001). Increasing the DTT concentration enhanced 6-TG recovery. The hydrolysis time used in the Lennard method (1 h) was not sufficient to achieve complete hydrolysis. CONCLUSIONS: The difference between 6-TGN concentrations measured by the two methods is attributable, at least in part, to differences in the extent of nucleotide hydrolysis. For optimization of thiopurine therapy, method-dependent therapeutic ranges are necessary, which precludes comparison of results from clinical studies derived with these methods. Efforts must therefore be made to standardize the analytical procedures for the determination of 6-TGN.

Acyl glucuronide drug metabolites: toxicological and analytical implications.

Although glucuronidation is generally considered a detoxification route of drug metabolism, the chemical reactivity of acyl glucuronides has been linked with the toxic properties of drugs that contain carboxylic acid moieties. It is now well documented that such metabolites can reach appreciable concentrations in blood. Furthermore, they are labile, undergo hydrolysis and pH-dependent intramolecular acyl migration to isomeric conjugates of glucuronic acid, and may react irreversibly with plasma proteins, tissue proteins, and with nucleic acids. This stable binding causes chemical alterations that are thought to contribute to drug toxicity either through changes in the functional properties of the modified molecules or through antigen formation with subsequent hypersensitivity and other immune reactions. Whereas in vitro data on the toxicity of acyl glucuronides have steadily accumulated, direct evidence for their toxicity in vivo is scarce. Acyl glucuronides display limited stability, which is dependent on pH, temperature, nature of the aglycon, and so on. Therefore, careful sample collection, handling, and storage procedures are critical to ensure generation of reliable pharmacologic and toxicologic data during clinical studies. Acyl glucuronides can be directly quantified in biologic specimens using chromatographic procedures. Their adducts with plasma or cell proteins can be determined after electrophoretic separation, followed by blotting. ELISA techniques have been used to assess the presence of antibodies against acyl glucuronide-protein adducts. This review summarizes the most recent evidence concerning biologic and toxicologic effects of acyl glucuronide metabolites of various drugs and discusses their relevance for drug monitoring. A critical evaluation of the available methodology is included.

Atypical pharmacokinetics and metabolism of mycophenolic acid in a young kidney transplant recipient with impaired renal function.

A juvenile, female renal transplant recipient suffered two acute rejection episodes: the first on posttransplant day 31 while taking cyclosporine, prednisone, and mycophenolate mofetil (MMF); and the second on posttransplant day 67, when she was taking tacrolimus, prednisone, and MMF. Dosage of MMF was initially started at 2 g/d (corresponding to 600 mg MMF/m(2) twice daily.), but was reduced to 250 mg/d to 500 mg/d after severe diarrhea and a paralytic ileus on posttransplant day 16. During therapy with tacrolimus, prednisone, and MMF, predose plasma mycophenolic acid (MPA) concentrations varied from 1.1 mg/L to 8.2 mg/L (median 3.0 mg/L). On posttransplant day 91, a 12-hour pharmacokinetic profile was obtained. The concentrations of MPA and its metabolites were determined with a validated high-performance liquid chromatography (HPLC) procedure. After oral MMF (250 mg) administration, the MPA concentration showed an atypical decline from a predose concentration of 6.0 mg/L to a value of 3.8 mg/L at 75 minutes postdose, and 3.4 mg/L at 6 hours postdose, before returning to 6.0 mg/L after 12 hours. The 12-hour area under the concentration-time curve (AUC) values for MPA and its major metabolite the phenolic glucuronide MPAG were 55.1 mg.h/L and 800 mg.h/L, respectively. An unusually high concentration (12-h AUC, 165 mg.h/L) of the phenolic glucose conjugate of MPA was found. The apparent renal clearance of MPAG was only 2.2 mL/min. Her creatinine clearance was 30 mL/min. MPAG clearances have been reported to range from approximately. 5.5 mL/min to 35 mL/min at a creatinine clearance of approximately 30 mL/min in renal transplant recipients. The authors' findings suggest that conjugation and clearance of MPA through the kidney is strongly impaired in this patient. The relatively high predose MPA concentrations could result from an enhanced enterohepatic circulation of MPA and its metabolites.

Highly effective reduction of C-reactive protein in patients with coronary heart disease by extracorporeal low density lipoprotein apheresis.

An association between C-reactive protein (CRP) and coronary heart disease (CHD) has been shown. CRP is present in atherosclerotic lesions, and there is increasing evidence that it may contribute to inflammation. Reduction of CRP concentrations otherwise considered normal may thus be of therapeutic value. Heparin-induced extracorporeal low density lipoprotein precipitation (HELP) is an established apheresis procedure to treat CHD patients with hypercholesterolemia. CRP concentrations were determined pre- and post-apheresis in 13 hypercholesterolemic CHD patients, during a total of 31 treatment procedures as well as in the interval between two treatments in six-patients using a high-sensitivity CRP assay. In addition, the effect of the HELP precipitation buffer on serum CRP concentrations was investigated in vitro. HELP treatment reduced CRP concentrations on average by 65%. The presence of CRP in the LDL precipitate of a patient was also confirmed by Western-blot analysis. In vitro experiments with serum samples revealed that CRP was partly co-precipitated with LDL. Greater fluctuation was observed in the post-apheresis concentrations of CRP compared with LDL. These results show that CRP can be very effectively lowered in CHD patients through the HELP system. This may further explain the stabilization and reduction of atherosclerotic plaques in hypercholesterolemic patients previously demonstrated with this treatment procedure.

Two-hour cyclosporine concentration determination: an appropriate tool to monitor neoral therapy?

Cyclosporine is a critical dose drug for which individualisation by therapeutic drug monitoring is indisputable. Current evidence suggests that a single concentration (C2) taken two hours after cyclosporine administration with the microemulsion formulation better predicts exposure and events than the trough concentration (C(0)), which is routinely used for adjusting the dosage of this drug. Studies have shown that the greatest calcineurin inhibition and the maximum inhibition of IL-2 production occur in the first 1 to 2 hours after dosing. These findings support the concept that the C2 level better reflects immunosuppressive efficacy than the trough concentration. Preliminary data from an outcome study in liver transplant recipients have shown that the incidence of biopsy proven moderate to severe acute rejection was significantly lower in patients managed by C2 monitoring compared with those monitored by C(0). The critical importance of achieving adequate cyclosporine exposure during the first 3 to 5 posttransplant days to prevent acute rejection has been documented in prospective studies with de novo renal and liver transplant recipients. Conversion of maintenance liver and heart transplant patients to C2 monitoring resulted in an amelioration of renal function. Time-dependent target values have been proposed for liver and renal transplant recipients. These require further prospective validation. For routine monitoring of C2 levels on-site validated dilution guidelines are necessary for most of the available immunoassays. C2 monitoring necessitates further organizational requirements which may be judged differently between transplant centers. In particular during the early posttransplant period C2 monitoring is a promising new option to make immunosuppressive therapy with the microemulsion formulation of cyclosporine safer and more efficient.